In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

From selection to caged aptamers: Identification of light-dependent ssDNA aptamers targeting cytohesin

Caged aptamers represent valuable tools for the spatiotemporal control of protein function by light. Here we describe a general route starting with the de novo selection process targeting cytohesin-1 and aiming at the synthesis of caged aptamers without the prior knowledge of detailed structural determinants of aptamer-target binding.     

Automated selection of anti-protein aptamers

The in vitro selection of nucleic acid binding species (aptamers) is frequently repetitive, time-consuming, and poorly adapted to high-throughput applications. We have adapted automated workstations to select anti-protein aptamers; as an example, we demonstrated the selection of anti-lysozyme aptamers that function as efficient inhibitors of cell lysis. The increases in throughput brought about by automation should potentiate the application of aptamer technology to the rapidly growing field of proteomics.     

In vitro selection of signaling aptamers

Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals.     

Direct selection of RNA beacon aptamers

A method for the direct selection of RNA molecules that can be easily converted into beacon aptamers is presented. Beacon aptamers are fluorescently labeled nucleic acids that signal the presence of a specific ligand through changes in fluorescence intensity. Typically, ligand binding causes an increase in fluorescence intensity by inducing a conformational change that separates a fluorophore/quencher pair. The method presented here simultaneously selects for ligand binding and induction of an appropriate conformational change. The method was tested by selecting RNA molecules that can detect the aminoglycoside antibiotic tobramycin. After 14 rounds of selection, two sequence families emerged. Upon conversion into beacon aptamers, representatives of the two selected sequence families specifically detected tobramycin, while a negative control RNA that did not survive the selection protocol did not function as a tobramycin beacon aptamer.     

In silico selection of RNA aptamers

In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer–ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude—significantly accelerating the experimental screening and selection of high-affinity aptamers.     

Visual detection of labeled oligonucleotides using visible-light-polymerization-based amplification

DNA biochip technology holds potential for highly parallel, rapid, and sensitive genetic diagnostic screening of target pathogens and disease biomarkers. A primary limitation involves a simultaneous, sequence-specific identification of low copy number target polynucleotides using a clinically appropriate detection methodology that implements only inexpensive detection instrumentation. Here, a rapid (20 min), nonenzymatic method of signal amplification utilizing surface-initiated photopolymerization is presented in glass microarray format. Visible light photoinitiators covalently coupled to streptavidin were used to bind biotin-labeled capture sequences. Amplification was achieved through subsequent contact with a monomer solution and the appropriate light exposure to generate 20-240-nm-thick hydrogel layers exclusively from spots containing the biotin-labeled DNA. An amplification factor of 10(6) to 10(7) was observed as well as a detectable response generated from as low as approximately 10(4) labeled oligonucleotides using minimal instrumentation, such as an optical microscope or CCD camera.     

Protein detection via direct enzymatic amplification of short DNA aptamers

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Here we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. Because both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 microM to 10 pM).     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

In vitro selection of hematoporphyrin binding DNA aptamers

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX.     

Methods for selection of aptamers to protein targets

Aptamers are synthetic single-stranded RNA or DNA molecules capable of specific binding to other target molecules. In this review, the main aptamer properties are considered and methods for selection of aptamers against various protein targets are described. Special attention is given to the methods for directed selection of aptamers, which allow one to obtain ligands with specified properties.     

The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers

A PCR method for uniform amplification of a random sequence DNA library is described. A combination of 1 M betaine and 5% DMSO improves the PCR amplification by increasing the ratio of full-length products to shortened products, which are a consequence of nonuniform amplification due to stable secondary structures in the templates. This method is expected to be beneficial for obtaining high-affinity aptamers with stable secondary structures.     

Conformational and chiral selection of oligonucleotides

In view of a better understanding of chiral selection of oligonucleotides, we have studied the hybridization of D- and L-CNA (cyclohexane nucleic acids) and D- and L-DNA, with chiral D-beta-homo-DNA and achiral PNA (peptide nucleic acids). PNA hybridizes as well with D-DNA, L-DNA as with D-beta-homo-DNA. The structure of the PNA x D-beta-homo-DNA complex is different from the PNA x DNA duplexes. D-CNA prefers D-DNA as hybridization partner, while L-CNA prefers D-beta-homo-DNA as hybridization partner. The conformation of the enantiomeric oligonucleotides D-CNA and L-CNA in the supramolecular complex with D-DNA and D-beta-homo-DNA, respectively, is different. These data may contribute to the confirmation of a hypothesis of the existence of achiral informative polymers as RNA predecessor, and to the understanding of homochirality of nucleic acids.     

In vitro selection and characterization of cellulose-binding DNA aptamers

Many nucleic acid enzymes and aptamers have modular architectures that allow them to retain their functions when combined with other nucleotide sequences. This modular function facilitates the engineering of RNAs and DNAs that have more complex functions. We sought to create new DNA aptamers that bind cellulose to provide a module for immobilizing DNAs. Cellulose has been used in a variety of applications ranging from coatings and films to pharmaceutical preparations, and therefore DNA aptamers that bind cellulose might enable new applications. We used in vitro selection to isolate aptamers from a pool of random-sequence DNAs and subjected two distinct clones to additional rounds of mutagenesis and selection. One aptamer (CELAPT 14) was chosen for sequence minimization and more detailed biochemical analysis. CELAPT 14 aptamer variants exhibit robust binding both to cellulose powder and paper. Also, an allosteric aptamer construct was engineered that exhibits ATP-mediated cellulose binding during paper chromatography.     

The selection of aptamers specific for membrane molecular targets

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.     

Semi-automated selection of DNA aptamers using magnetic particle handling

We have developed a semi-automatic selection procedure for DNA aptamers. Employing a robotic workstation for magnetic particle handling, this method allows for a fast, reproducible, and parallelized selection of DNA aptamers. The selection protocol is designed to provide high flexibility and versatility in terms of choice of buffers and reagents, as well as stringency of selection. Using this procedure, we have successfully isolated ligand-specific, high-affinity DNA aptamers.     

In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated K_D values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.     

In vitro selection of DNA aptamers that bind L-tyrosinamide

We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design.     

Altering molecular recognition of RNA aptamers by allosteric selection

In a continuing effort to explore structural and functional dynamics in RNA catalysis, we have created a series of allosteric hammerhead ribozymes that are activated by theophylline. Representative ribozymes exhibit greater than 3000-fold activation upon effector-binding and cleave with maximum rate constants that are equivalent to the unmodified hammerhead ribozyme. In addition, we have evolved a variant allosteric ribozyme that exhibits an effector specificity change from theophylline to 3-methylxanthine. Molecular discrimination between the two effectors appears to be mediated by subtle conformational differences that originate from displacement of the phosphodiester backbone near the effector binding pocket. These findings reveal the importance of abstruse aspects of molecular recognition by nucleic acids that are likely to be unapproachable by current methods of rational design.